Immunogenetics

Hollenbach, Jill; Madbouly, Abeer; Gragert, Loren; Vierra-Green, Cynthia; Flesch, Susan; Spellman, Stephen; Begovich, Ann; Noreen, Harriet; Trachtenberg, Elizabeth; Williams, Tom; Yu, Neng; Shaw, Bronwen; Fleischhauer, Katharina; Fernandez-Vina, Marcelo; Maiers, Martin

doi: 10.1007/s00251-012-0615-3pmid: 22526601

Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context.

Flajnik, Martin; Tlapakova, Tereza; Criscitiello, Michael; Krylov, Vladimir; Ohta, Yuko

doi: 10.1007/s00251-012-0616-2pmid: 22488247

The B7 family of genes is essential in the regulation of the adaptive immune system. Most B7 family members contain both variable (V)- and constant (C)-type domains of the immunoglobulin superfamily (IgSF). Through in silico screening of the Xenopus genome and subsequent phylogenetic analysis, we found novel genes belonging to the B7 family, one of which is the recently discovered B7H6. Humans and rats have a single B7H6 gene; however, many B7H6 genes were detected in a single large cluster in the Xenopus genome. The B7H6 expression patterns also varied in a species-specific manner. Human B7H6 binds to the activating natural killer receptor, NKp30. While the NKp30 gene is single-copy and maps to the MHC in most vertebrates, many Xenopus NKp30 genes were found in a cluster on a separate chromosome that does not harbor the MHC. Indeed, in all species so far analyzed from sharks to mammals, the number of NKp30 and B7H6 genes correlates well, suggestive of receptor-ligand co-evolution. Furthermore, we identified a Xenopus-specific B7 homolog (B7HXen) and revealed its close linkage to B2M, which we have demonstrated previously to have been originally encoded in the MHC. Thus, our study provides further proof that the B7 precursor was included in the proto MHC. Additionally, the comparative analysis revealed a new B7 family member, B7H7, which was previously designated in the literature as an unknown gene, HHLA2.

Belyaev, Nikolai; Biró, Judit; Athanasakis, Dimitrios; Fernandez-Reyes, Delmiro; Potocnik, Alexandre

doi: 10.1007/s00251-012-0620-6pmid: 22581009

T cell development constitutes a multistage process allowing the dissection of events resulting in cellular commitment and functional specification in a specialized microenvironment. This process is guided by the appropriate expression of regulatory genetic factors like transcriptional activators or repressors which are, in part, dependent on instructive signals of the microenvironment. To date, it remains unclear whether exactly the same genetic mechanism acts in adult compared to fetal T cell development. In order to directly compare T cell commitment during adult and fetal differentiation, we isolated subsequent stages of intrathymic subpopulations starting with early canonical T cell progenitors up to irreversibly committed T cell precursors. The genome-wide analysis revealed several distinct gene clusters with a specific pattern of gene regulation for each subset. The largest cluster contained genes upregulated after transition through the most primitive pool into the next transitory population with a consistently elevated expression of elements associated with ongoing T cell fate specification, like Gata3 and Tcf7, in fetal progenitors. Furthermore, adult and fetal T cell progenitors occupied distinct “transcriptional territories” revealing a precise land map of the progression to final T cell commitment operating in different developmental windows. The presence and/or elevated expression of elements associated with an ongoing establishment of a T cell signature in the most primitive fetal subset is highly suggestive for an extrathymic initiation of T cell specification and underlines the fundamental differences in fetal versus adult lymphopoiesis.

Blancher, Antoine; Aarnink, Alice; Tanaka, Keiko; Ota, Masao; Inoko, Hidetoshi; Yamanaka, Hisashi; Nakagawa, Hiroshi; Apoil, Pol-André; Shiina, Takashi

doi: 10.1007/s00251-012-0613-5pmid: 22790512

The cynomolgus macaque (Macaca fascicularis) is currently used as an animal model in various fields of immunology especially in the development of innovative vaccines for the prevention and treatment of infectious diseases. The polymorphism of the major histocompatibility complex (MHC) influences the development of adaptive immune responses, and it is crucial to characterize the polymorphism of cynomolgus MHC genes. Among all macaque species, the cynomolgus macaque has the most diversified geographical area encompassing continental and insular populations. By the study of a large sample of animals from the Philippines (N = 359), we have characterized 20 DRB haplotypes. The DRB genotyping was performed by denaturing gradient gel electrophoresis (DGGE) sequencing of exon 2 and was confirmed by polymerase chain reaction–sequence-specific oligonucleotide. The DRB and DRA cDNA of 126 animals were characterized by cloning and sequencing. By means of DGGE sequencing, we characterized the polymorphism of genomic DRB exon 2 in three other cynomolgus macaque population samples (Java, Vietnam, and Mauritius), and we discuss about the origin of the founders of the Mauritian and the Filipino cynomolgus macaque populations.

Groot, Natasja; Otting, Nel; Robinson, James; Blancher, Antoine; Lafont, Bernard; Marsh, Steven; O’Connor, David; Shiina, Takashi; Walter, Lutz; Watkins, David; Bontrop, Ronald

doi: 10.1007/s00251-012-0617-1

Lundgren, Alyssa; Kim, Sharon; Stadnisky, Michael; Brown, Michael

doi: 10.1007/s00251-012-0630-4pmid: 22752191

Ly49G and H-2 class I Dk molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I Dk disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F2 and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.

Parra, Zuly; Miller, Robert

doi: 10.1007/s00251-012-0621-5pmid: 22592501

The access to whole genome sequences has provided the opportunity to study the evolution and organization of immunologically related genes on a large scale. The genes encoding the T cell receptor (TCR) α and δ chains are part of a complex locus that has shown remarkable conserved organization across different amniote lineages. In this study we have examined and annotated the TCRα/δ locus in chicken (Gallus gallus) and compared it to that of zebra finch (Taeniopygia guttata) and other avian species using the current available genome data. We also analyzed the expressed chicken TCRα/δ transcript repertoire and compared it with that previously described for zebra finch. The analyses conducted in this study show that the TCRα/δ locus in birds has undergone major rearrangements and expansion of the germ line repertoire in chicken, compared to zebra finch. A major expansion of the chicken variable gene repertoire appears to be driven by selection for genes from a limited number of subgroups.

Wang, Xinxin; Parra, Zuly; Miller, Robert

doi: 10.1007/s00251-012-0626-0pmid: 22684248

A VpreB surrogate light (SL) chain was identified for the first time in a marsupial, the opossum Monodelphis domestica. Comparing the opossum VpreB to homologues from eutherian (placental mammals) and avian species supported the marsupial gene being VpreB3. VpreB3 is a protein that is not known to traffic to the cell surface as part of the pre-B cell receptor. Rather, VpreB3 associates with nascent immunoglobulin chains in the endoplasmic reticulum. Homologues of other known SL chains VpreB1, VpreB2, and λ5, which are found in eutherian mammals, were not found in the opossum genome, nor have they been identified in the genomes of nonmammals. VpreB3 likely evolved from earlier gene duplication, independent of that which generated VpreB1 and VpreB2 in eutherians. The apparent absence of VpreB1, VpreB2, and λ5 in marsupials suggests that an extracellular pre-B cell receptor containing SL chains, as it has been defined in humans and mice, may be unique to eutherian mammals. In contrast, the conservation of VpreB3 in marsupials and its presence in nonmammals is consistent with previous hypotheses that it is playing a more primordial role in B cell development.